Major histocompatibility (MHC) Class I antigens are highly polymorphic. There are numerous alleles. Each molecule contains multiple amino acid substitutions; this polymorphism is required for recognition of viral pathogens by T cells. Our goal has been to delineate structure-function relationships of the Class I antigen. To this end DNA sequence encoding a mouse Class I gene is modified in vitro to examine the functions associated with the modification. In the past year oligonucleotide directed mutagenesis has been employed to introduce single amino acid substitutions into the H-2Ld gene. The following mutations were generated: (1) The S-S linkage in the 2nd domain was disrupted by replacing Cys with Ser(2). The glycosylation site was removed from the first external domain. (3) Phe at position 116 was changed to Tyr to replace the Ld residue with the Kb type amino acid. The mutant genes were transferred into L cells by DNA mediated gene transfer and the products of the mutants were examined for surface expression, antigenicity by antibody binding, and for T cell reactivity. The mutant gene with the disrupted disulfide bridge was expressed on cell surface, indicating that the disulfide bridge is not required for surface expression of the antigens even though it is highly conserved throughout mammalian species. However, numerous antigenic determinants for T cells and for antibodies were no longer found in the mutant antigen, indicating that the tertiary structure dictated by the disulfide bridge is important for the function of the Class I antigen. The mutants with the altered glycosylation site and that with altered amino acid at position 116 expressed antigens identical to those of the wild type gene products. Studies of domain specificities of numerous new monoclonal antibodies reacting with the H-2Dd antigen has been completed. Comparisons of amino acid sequences among different H-2 antigens including Dd led us to propose distinct amino acid positions to be the antigenic sites. Based on these predictions systematic introduction of mutagenesis is planned for the H-2Dd gene to identify the functional sites of the antigen.